FIG. 2.

Effect of mutations L143A and L178A on Lb^pro self-processing at the L/VP4 junction containing the substitution L200F. RRLs were incubated with or without 10 ng/μl of mRNAs encoding the wild type (Lb^proVP4/VP2 wt) (A), Lb^proVP4/VP2 L200F (B), Lb^proVP4/VP2 L143A L200F (C), or Lb^proVP4/VP2 L178A L200F (D). Ten-microliter samples were taken at the indicated time points, and protein translation was terminated by placing the samples on ice, followed by addition of unlabeled methionine and cysteine to final concentrations of 2 mM and Laemmli sample buffer. Ten-microliter aliquots were analyzed on a 17.5% sodium dodecyl sulfate-polyacrylamide gel, followed by fluorography. The negative controls (N) were incubated for 60 min without RNA. The amino acid sequences at the Lb^pro/VP4 junction are given. The positions of uncleaved Lb^proVP4/VP2 and cleavage products Lb^pro and VP4/VP2 are marked. Protein standards (in kDa) are shown on the left.